Project Summary Botulinum neurotoxin serotype A (BoNT/A) is the most lethal known human poison, exhibiting a potency 1011 times that of cyanide (LD50 ~ 1-5 ng/kg). In addition to this remarkable toxicity, the ease of BoNT/A production and the absence of effective post-exposure interventions have prompted the Centers for Disease Control (CDC) to classify BoNT/A as a biodefense threat of the highest risk to national security. Although immunotherapies exist to protect against BoNT/A toxicity, their effectiveness is lost once the toxin is internalized into neuronal cells (<12 h post-exposure). Compounding this fact, BoNT/A persists intraneuronally on the order of months to >1 year in its toxic form. Small molecules offer the only possibility of inhibiting intraneuronal BoNT/A as these compounds can cross neuronal membranes; however, the development of clinically-relevant therapies has been limited by the short half-lives of these inhibitors, which are only a fraction of the timescale of BoNT/A persistence. In this proposal, we present two pharmacotherapeutic strategies for overcoming BoNT/A persistence in neurons. In the first approach, ubiquitination and proteasomal degradation of BoNT/A can be induced using a bifunctional small molecule known as a proteolysis targeting chimera, or PROTAC. These molecules rely on conjugating a vetted BoNT/A antagonist to an E3 ubiquitin ligase-recruiting ligand. In the cell, these PROTACs tether BoNT/A to the cell?s protein degradation machinery. In the second approach, we will convert known zinc chelating inhibitors of BoNT/A into bifunctional compounds that also covalently engage cysteine 165 of the BoNT/A active site. Covalent modification of this residue will permanently modify the conformation of the active site, inhibiting BoNT/A catalysis and thus, toxicity. To validate these strategies, we will design and synthesize PROTACs (Aim 1) and covalent inhibitors (Aim 2) based on previously reported hydroxamate BoNT/A inhibitors developed by our laboratory. After synthesis, we will extensively evaluate these compounds in in vitro and cellular assays for BoNT/A inhibition. The proposed work will help to achieve our long-term goal of delivering a clinically-relevant pharmacotherapeutic strategy for treating post-exposure BoNT/A intoxication. The objective here is to deliver the first small molecules that effectively overcome BoNT/A persistence in the neuronal compartment. This work is important because we will deliver tools to probe mechanisms of BoNT/A persistence; in addition to, offering novel strategies in BoNT/A therapeutic design. Upon completion of this proposal, the most efficacious and ?drug-like? molecules will be prime for evaluation in relevant in vivo models. This goal is aligned with the mission of the NIAID to support research aimed at developing state-of-the-art treatments for biowarfare agents.